The human papillomavirus (HPV) E6 protein is needed for viral replication. Infection with ?high risk? HPV types can progress to pre-malignant lesions called dysplasias, which over a period of years, can eventuate in invasive and metastatic epithelial malignancies. HPV E6 proteins bind to the ubiquitin ligase E6AP and this complex targets p53 and other E6 binding proteins for proteasome mediated destruction. This E6-dependent loss of p53 enables HPV to bypass host cell defenses and facilitates activation of the cell cycle. E6AP and other E6 interacting proteins utilize a charged leucine ?-helical LxxLL peptide motif to bind to E6. Using molecular modeling based on the structure of this E6AP motif, we previously identified a series of novel flavone like molecules that inhibit HPV-16 E6 binding to E6AP. Exposure of cervical cancer cells to these compounds led to increases inp53 and p21Cip1/Waf1 proteins and decreased proliferation of HPV expressing cell lines. We used computational modeling to predict where these compounds bind onto the recently described high-resolution three-dimensional co-crystal structure of HPV-16 E6 with an LxxLL peptide. The highest scoring fits placed the flavone inhibitors in a hydrophobic pocket that forms molecular bonds with leucines in the E6AP binding pocket and a series of flanking arginines of E6. These data support our structure-based screening and compound selection based on inhibition of E6 association with E6AP. Importantly, LxxLL peptide binding induces an allosteric change in the E6 protein needed for entry of p53 into the complex. Our inhibitors may interfere with this conformational change in E6 and/or its protein-protein interaction with p53. Flavonoid small molecules inhibit HPV E6 but have poor aqueous solubility and unfavorable drug metabolism properties and thus are suboptimal as therapeutics. Additionally, development of small molecule HPV E6 inhibitor therapeutics will require a proprietary compound class. We took advantage of the flavone- based structure-activity relationships and modified the core structure to establish a novel and highly druggable chemical series. Pilot studies prove we can alter the scaffold and retain E6 inhibitory activity. While less potent than the flavones, this SBIR grant will allow us to rapidly increase potency using established assays of competitive inhibition of E6AP binding, E6 thermo-stabilization, and p53 degradation in vitro and in cells. A focused series of mutated E6 proteins that disrupt the hydrophobic and charged surfaces of the E6 binding pocket let us explore the binding interface between E6 and inhibitory compounds. We have observed some point mutations disrupt the E6-E6AP interaction but do not alter compound binding to E6 and, conversely, some mutants retain E6AP binding but restrict compound association. These data substantiate our positioning of compounds on the E6 structure and instruct design of more specific and potent HPV E6 inhibitors. We have shown we can select and synthesize inhibitors of the E6-E6AP interaction and thus potentially, a novel treatment for the millions afflicted with pre-malignant and malignant HPV associated cancers.